Development And Functional Studies Of Yak-derived Collagen Peptides

According to the 7th national census data released in 2021,China’s population aged 60 and above has reached 264 million,accounting for 18.7%of the total population.The aging of the population has led to an increase in the incidence and amount of osteoporosis in our nation year by year.However,the side effects of the current therapeutic drugs in the market are relatively big.Thereby the prevention of osteoporosis has great practical significance and scientific research value.The collagen peptides obtained by enzymatic hydrolysis of yak bone collagen are rich in essential amino acids which can be absorbed easily by the human body,which is one of the main sources of collagen nutrition products on the market.Clinical trials have shown that collagen hydrolyzate has antioxidant and anti-osteoporotic effects.However,the constitute of hydrolyzed bovine collagen products is complex,the functional active ingredients are unknown,so the mechanism is elusive.Therefore,developing bioactive peptides from hydrolysates with a clear mechanism will help to exert higher application value of collagen peptides,which can be applied to the fields of health food or even drug development in the future.In this work,the crude yak bone collagen was enzymatically hydrolyzed by the collagenase previously excavated in our laboratory.Afterward,the enzymatic hydrolysate was separated and collected.The peptide sequence was identified by RPHPLC-MS/MS.The scavenging experiments of ABTS·radical,DPPH·radical,hydroxyl radical and superoxide anion radical were applied to screen peptides of their in vitro antioxidant activities.Treated on Caenorhabditis elegans as an animal model,the anti-oxidative effects of active peptides in vivo,as well as their mechanism,were studied.In addition,functional peptides that promote osteogenesis were developed using the MC3T3-E1 cell model.This study provides not only active peptide candidates but also a theoretical basis for food or drug development in the field of anti-aging and osteoporosis prevention.The main work of this paper includes the following four parts:(1)The enzymatic hydrolyzed product was obtained by decomposing bovine crude collagen with collagenase from Bacillus cereus,the components of the enzymatic hydrolysis product were separated and identified in mutiple times,then a library containing 245 collagen and non-collagen peptides was obtained.The molecular weight distribution of the peptides in the library was from 500 to 2800 Da,of which 180 peptides was below 1500 Da,accounting for 73.47%of the entire library.Analyzed after contrasting,15 selected peptides were synthesizedfrom the library for in vitro activity validation.The in vitro antioxidant test showed that 13 water-soluble peptides had some antioxidant activity.In the DPPH·radical and superoxide anion free radical scavenging tests,the activity of every single peptide varies greatly.In conclusion,peptides UU1 and KC7 have good scavenging activities in all free radical scavenging tests in vitro,showing relatively comprehensive performance.(2)Using C.elegans as an animal model,at the same peptide concentration of 0.1 mg/mL,UU1 and KC7 were studied for their effects on lifespan in normal circumstances,thermal shock,locomotory behavior,reproductive capacity,as well as the accumulation of ROS and MDA.The activities of two antioxidant enzymes,superoxide dismutase(SOD)and catalase(CAT)were also tested.Furthermore,the respective regulatory mechanisms of these two peptides were studied to understand their regulation of genes related to antioxidant pathways.The results displayed that the peptide UU1 had a slight effect on prolonging the lifespan of nematodes,it also improved the reproductive capacity,the thermal resistances.It not only significantly enhanced the activities of SOD and CAT,but also reduced the levels of MDA and ROS,and up-regulated the transcription of genes daf-16,sir-2.1,sod-3 and skn-1.In contrast,KC7 had better effects on prolonging the lifespan of nematodes enhancing the ability of nematodes to resist heat stress,meanwhile,it prompted the nematodes to produce more offspring,even shortening the egg-laying period.Compared with the control,the 0.1 mg/mL KC7 treatment group reduced the accumulation of ROS in nematodes by 17.89%,the CAT activity significantly increased by 101.80%.The up-regulation of daf16,sod-3,skn-1 transcript levels by KC7 was almost double that of the UU1 group,but the increase of hsf-1transcript level was only observed in the KC7 group.3)In this paper,the effects of peptide UU1 on proliferation,cell cycle,differentiation,apoptosis,and mineralization of MC3T3-E1 cells were investigated.The results showed that the polypeptide UU1 could simultaneously activate the gene transcription and protein expression of Akt accompanied by β-catenin signaling pathways in MC3T3-E1 cells.UU1 also promoted cell proliferation along with differentiation.It increased the expression of alkaline phosphatase(ALP),Cyclin Dl,Runx2,Collagen I,and Ocn proteins.In addition,with the increasing concentration of UU1,the expression ratio of Bax/Bcl-2 protein in MC3T3-E1 cells also decreased.UU1 exhibited good osteogenic activity to accelerate the proliferation,differentiation,and mineralization of MC3T3-E1 cells.What is more,UU1 may even help delay cell apoptosis.Taken together,the performance of UU1 was more prominent at concentrations of 25 μg/mL along with 50 μg/mL.UU1 has great potential for application in bone protection and even the effective treatment of osteoporosis.(4)The osteopromoting activity of peptide KC7 was studied in MC3T3-E1 cells.Compared with the control group,KC7 promoted cell proliferation.Under the treatment of 12.5 μg/mL KC7,the intracellular ALP enzyme activity coupled with the expression of the ALP gene increased simultaneously.At two lower concentrations,the expression of　important differentiation-related genes Runx2,Osterix,Ocn,Col2,etc were increased.All three concentrations of KC7 increased the amount of Runx2 and Collagen I proteins to facilitate cell differentiation,as well as mineralization.In addition,KC7 regulates βcatenin in a concentration-dependent manner.However,the increase of Ocn protein expression,the phosphorylation of Akt and the downregulation of the Bax/Bcl2 ratio only appeared at 12.5 μg/mL KC7 treatment.Therefore,KC7 is a peptide with osteogenic activity,it seems to be more beneficial in accelerating the proliferation along with the cell cycle of MC3T3-E1 cells and delaying apoptosis at low concentrations.